Intrathecal delivery of recombinant adeno-associated virus 9

ABSTRACT

The present invention relates to Adeno-associated virus type 9 methods and materials useful for intrathecal delivery of polynucleotides. Use of the methods and materials is indicated, for example, for treatment of lower motor neuron diseases such as SMA and ALS as well as Pompe disease and lysosomal storage disorders. It is disclosed that administration of a non-ionic, low-osmolar contrast agent, together with a rAAV9 vector for the expression of Survival Motor Neuron protein, improves the survival of SMN mutant mice as compared to the administration of the expression vector alone.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/678,458 filed Aug. 1, 2012, which is incorporated by reference in itsentirety herein.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under RC2 NS69476-01awarded by the National Institutes of Health (NIH). The Government hascertain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a sequence listing incomputer-readable form submitted concurrently herewith and identified asfollows: ASCII text file named “47099PCT_SeqListing.txt”, 8,954 bytes,created 31 Jul. 2013.

FIELD OF THE INVENTION

The present invention relates to Adeno-associated virus type 9 methodsand materials useful for intrathecal delivery (i.e., delivery into thespace under the arachnoid membrane of the brain or spinal cord) ofpolynucleotides. Use of the methods and materials is indicated, forexample, for treatment of lower motor neuron diseases such as SMA andALS as well as Pompe disease and lysosomal storage disorders.

BACKGROUND

Large-molecule drugs do not cross the blood-brain-barrier (BBB) and 98%of small-molecules cannot penetrate this barrier, thereby limiting drugdevelopment efforts for many CNS disorders [Pardridge, W. M. Nat RevDrug Disco 1: 131-139 (2002)]. Gene delivery has recently been proposedas a method to bypass the BBB [Kaspar, et al., Science 301: 839-842(2003)]; however, widespread delivery to the brain and spinal cord hasbeen challenging. The development of successful gene therapies for motorneuron disease will likely require widespread transduction within thespinal cord and motor cortex. Two of the most common motor neurondiseases are spinal muscular atrophy (SMA) and amyotrophic lateralsclerosis (ALS), both debilitating disorders of children and adults,respectively, with no effective therapies to date. Recent work in rodentmodels of SMA and ALS involves gene delivery using viruses that areretrogradely transported following intramuscular injection [Kaspar etal., Science 301: 839-842 (2003); Azzouz et al., J Clin Invest 114:1726-1731 (2004); Azzouz et al., Nature 429: 413-417 (2004); Ralph etal., Nat Med 11: 429-433 (2005)]. However, clinical development may bedifficult given the numerous injections required to target thewidespread region of neurodegeneration throughout the spinal cord,brainstem and motor cortex to effectively treat these diseases. AAVvectors have also been used in a number of recent clinical trials forneurological disorders, demonstrating sustained transgene expression, arelatively safe profile, and promising functional responses, yet haverequired surgical intraparenchymal injections [Kaplitt et al., Lancet369: 2097-2105 (2007); Marks et al., Lancet Neurol 7: 400-408 (2008);Worgall et al., Hum Gene Ther (2008)].

SMA is an early pediatric neurodegenerative disorder characterized byflaccid paralysis within the first six months of life. In the mostsevere cases of the disease, paralysis leads to respiratory failure anddeath usually by two years of age. SMA is the second most commonpediatric autosomal recessive disorder behind cystic fibrosis with anincidence of 1 in 6000 live births. SMA is a genetic disordercharacterized by the loss of lower motor neurons (LMNs) residing alongthe length of the entire spinal cord. SMA is caused by a reduction inthe expression of the survival motor neuron (SMN) protein that resultsin denervation of skeletal muscle and significant muscle atrophy. SMN isa ubiquitously expressed protein that functions in U snRNP biogenesis.

In humans there are two very similar copies of the SMN gene termed SMN1and SMN2. The amino acid sequence encoded by the two genes is identical.However, there is a single, silent nucleotide change in SMN2 in exon 7that results in exon 7 being excluded in 80-90% of transcripts fromSMN2. The resulting truncated protein, called SMNΔ7, is less stable andrapidly degraded. The remaining 10-20% of transcript from SMN2 encodesthe full length SMN protein. Disease results when all copies of SMN1 arelost, leaving only SMN2 to generate full length SMN protein.Accordingly, SMN2 acts as a phenotypic modifier in SMA in that patientswith a higher SMN2 copy number generally exhibit later onset and lesssevere disease.

Therapeutic approaches for SMA have mainly focused on developing drugsfor increasing SMN levels or enhancing residual SMN function. Despiteyears of screening, no drugs have been fully effective for increasingSMN levels as a restorative therapy. A number of mouse models have beendeveloped for SMA. See, Hsieh-Li et al., Nature Genetics, 24 (1): 66-70(2000); Le et al., Hum. Mol. Genet., 14 (6): 845-857 (2005); Monani etal., J. Cell. Biol., 160 (1): 41-52 (2003) and Monani et al., Hum. Mol.Genet., 9 (3): 333-339 (2000). A recent study express a full length SMNcDNA in a mouse model and the authors concluded that expression of SMNin neurons can have a significant impact on symptoms of SMA. SeeGavrilina et al., Hum. Mol. Genet., 17(8):1063-1075 (2008).

ALS is another disease that results in loss of muscle and/or musclefunction. First characterized by Charcot in 1869, it is a prevalent,adult-onset neurodegenerative disease affecting nearly 5 out of 100,000individuals. ALS occurs when specific nerve cells in the brain andspinal cord that control voluntary movement gradually degenerate. Withintwo to five years after clinical onset, the loss of these motor neuronsleads to progressive atrophy of skeletal muscles, which results in lossof muscular function resulting in paralysis, speech deficits, and deathdue to respiratory failure.

The genetic defects that cause or predispose ALS onset are unknown,although missense mutations in the SOD-1 gene occurs in approximately10% of familial ALS cases, of which up to 20% have mutations in the geneencoding Cu/Zn superoxide dismutase (SOD1), located on chromosome 21.SOD-1 normally functions in the regulation of oxidative stress byconversion of free radical superoxide anions to hydrogen peroxide andmolecular oxygen. To date, over 90 mutations have been identifiedspanning all exons of the SOD-1 gene. Some of these mutations have beenused to generate lines of transgenic mice expressing mutant human SOD-1to model the progressive motor neuron disease and pathogenesis of ALS.

SMA and ALS are two of the most common motor neuron diseases. Recentwork in rodent models of SMA and ALS has examined treatment by genedelivery using viruses that are retrogradedly transported followingintramuscular injection. See Azzouz et al., J. Clin. Invest., 114:1726-1731 (2004); Kaspar el al., Science, 301: 839-842 (2003); Azzouz elal., Nature, 429: 413-417 (2004) and Ralph et al., Nature Medicine, 11:429-433 (2005). Clinical use of such treatments may be difficult giventhe numerous injections required to target neurodegeneration throughoutthe spinal cord, brainstem and motor cortex.

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thesingle-stranded DNA genome of which is about 4.7 kb in length including145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequenceof the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., JVirol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol,75: 3385-3392 (1994). Cis-acting sequences directing viral DNAreplication (rep), encapsidation/packaging and host cell chromosomeintegration are contained within the ITRs. Three AAV promoters (namedp5, p19, and p40 for their relative map locations) drive the expressionof the two AAV internal open reading frames encoding rep and cap genes.The two rep promoters (p5 and p19), coupled with the differentialsplicing of the single AAV intron (at nucleotides 2107 and 2227), resultin the production of four rep proteins (rep 78, rep 68, rep 52, and rep40) from the rep gene. Rep proteins possess multiple enzymaticproperties that are ultimately responsible for replicating the viralgenome. The cap gene is expressed from the p40 promoter and it encodesthe three capsid proteins VP1, VP2, and VP3. Alternative splicing andnon-consensus translational start sites are responsible for theproduction of the three related capsid proteins. A single consensuspolyadenylation site is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, CurrentTopics in Microbiology and Immunology, 158: 97-129 (1992).

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells, for example, in gene therapy. AAVinfection of cells in culture is noncytopathic, and natural infection ofhumans and other animals is silent and asymptomatic. Moreover, AAVinfects many mammalian cells allowing the possibility of targeting manydifferent tissues in vivo. Moreover, AAV transduces slowly dividing andnon-dividing cells, and can persist essentially for the lifetime ofthose cells as a transcriptionally active nuclear episome(extrachromosomal element). The AAV proviral genome is infectious ascloned DNA in plasmids which makes construction of recombinant genomesfeasible. Furthermore, because the signals directing AAV replication,genome encapsidation and integration are contained within the ITRs ofthe AAV genome, some or all of the internal approximately 4.3 kb of thegenome (encoding replication and structural capsid proteins, rep-cap)may be replaced with foreign DNA such as a gene cassette containing apromoter, a DNA of interest and a polyadenylation signal. The rep andcap proteins may be provided in trans. Another significant feature ofAAV is that it is an extremely stable and hearty virus. It easilywithstands the conditions used to inactivate adenovirus (56° to 65° C.for several hours), making cold preservation of AAV less critical. AAVmay even be lyophilized. Finally, AAV-infected cells are not resistantto superinfection.

Multiple serotypes of AAV exist and offer varied tissue tropism. Knownserotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10 and AAV11. AAV9 is described in U.S. Pat. No.7,198,951 and in Gao et al., J. Virol., 78: 6381-6388 (2004). Advancesin the delivery of AAV6 and AAV8 have made possible the transduction bythese serotypes of skeletal and cardiac muscle following simple systemicintravenous or intraperitoneal injections. See Pacak et al., Circ. Res.,99(4): 3-9 (1006) and Wang et al., Nature Biotech., 23(3): 321-8 (2005).The use of AAV to target cell types within the central nervous system,though, has required surgical intraparenchymal injection. See, Kaplittet al., supra; Marks et al., supra and Worgall et al., supra.

There thus remains a need in the art for methods and vectors fordelivering polynucleotides to the central nervous system.

SUMMARY

The present invention provides methods and materials useful forintrathecal delivery of polynucleotides to the central nervous systemusing recombinant a recombinant AAV9 (rAAV9) as a vector.

More specifically, the invention provides methods of delivering apolynucleotide to the central nervous system of a patient in needthereof comprising intrathecal delivery of rAAV9 and a non-ionic,low-osmolar contrast agent to the patient, wherein the rAAV9 comprises aself-complementary genome including the polynucleotide. Thepolynucleotide is delivered to, for example, the brain, the spinal cord,a glial cell, an astrocyte and/or a lower motor neuron. The non-ionic,low-osmolar contrast agent is, for example, iobitridol, iohexol,iomeprol, iopamidol, iopentol, iopromide, ioversol or ioxilan. In someembodiments, the polynucleotide is a survival motor neuron (SMN)polynucleotide.

The invention also provides methods of treating a neurological diseasein a patient in need thereof comprising intrathecal delivery of a rAAV9and a non-ionic, low-osmolar contrast agent to the patient, wherein therAAV9 comprises a self-complementary genome including a therapeuticpolynucleotide. The neurological disease is, for example, aneurodegenerative disease such as spinal muscular atrophy or amyotrophiclateral sclerosis. The therapeutic polynucleotide is, for example, anSMN polynucleotide. The SMN polynucleotide is delivered, for example, tothe brain, the spinal cord, a glial cell, an astrocyte and/or a lowermotor neuron. The non-ionic, low-osmolar contrast agent is, for example,iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversolor ioxilan.

DETAILED DESCRIPTION

Therefore, in one aspect, the invention provides a method forintrathecal delivery of a polynucleotide to the central nervous systemof a patient comprising administering a rAAV9 with a genome includingthe polynucleotide. In some embodiments, a non-ionic, low-osmolarcontrast agent is also administered to the patient. The non-ionic,low-osmolar contrast agent increases transduction of target cells in thecentral nervous system of the patient. In some embodiments, the rAAV9genome is a self-complementary genome. In other embodiments, the rAAV9genome is a single-stranded genome.

In some embodiments, the polynucleotide is delivered to brain. Areas ofthe brain contemplated for delivery include, but are not limited to, themotor cortex and the brain stem. In some embodiments, the polynucleotideis delivered to the spinal cord. In some embodiments, the polynucleotideis delivered to a lower motor neuron. Embodiments of the inventionemploy rAAV9 to deliver polynucleotides to nerve and glial cells. Insome embodiments, the glial cell is a microglial cell, anoligodendrocyte or an astrocyte. In some embodiments, the rAAV9 is usedto deliver a polynucleotide to a Schwann cell.

Use of methods and materials of the invention is indicated, for example,for treatment of lower motor neuron diseases such as SMA and ALS as wellas Pompe disease, lysosomal storage disorders, Glioblastoma multiformeand Parkinson's disease. Lysosomal storage disorders include, but arenot limited to, Activator Deficiency/GM2 Gangliosidosis,Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storagedisease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease,Fabry disease, Farber disease, Fucosidosis, Galactosialidosis, GaucherDisease (Type I, Type II, Type III), GM1 gangliosidosis (Infantile, Lateinfantile/Juvenile, Adult/Chronic), I-Cell disease/Mucolipidosis II,Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile HexosaminidaseA Deficiency, Krabbe disease (Infantile Onset, Late Onset),Metachromatic Leukodystrophy, Mucopolysaccharidoses disorders(Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, MPSI Hurler Syndrome,MPSI Scheie Syndrome, MPS I Hurler-Scheie Syndrome, MPS II Huntersyndrome, Sanfilippo syndrome Type A/MPS Ill A, Sanfilippo syndrome TypeB/MPS III B, Sanfilippo syndrome Type C/MPS III C, Sanfilippo syndromeType D/MPS III D, Morquio Type A/MPS IVA, Morquio Type B/MPS IVB, MPS IXHyaluronidase Deficiency, MPS VI Maroteaux-Lamy, MPS VII Sly Syndrome,Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV),Multiple sulfatase deficiency, Niemann-Pick Disease (Type A, Type B,Type C), Neuronal Ceroid Lipofuscinoses (CLN6 disease (Atypical LateInfantile, Late Onset variant, Early Juvenile),Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Finnish Variant LateInfantile CLN5, Jansky-Bielschowsky disease/Late infantile CLN2/TPP1Disease, Kufs/Adult-onset NCL/CLN4 disease, Northern Epilepsy/variantlate infantile CLN8, Santavuori-Haltia/Infantile CLN1/PPT disease,Beta-mannosidosis, Pompe disease/Glycogen storage disease type II,Pycnodysostosis, Sandhoff Disease/Adult Onset/GM2 Gangliosidosis,Sandhoff Disease/GM2 gangliosidosis—Infantile, Sandhoff Disease/GM2gangliosidosis—Juvenile, Schindler disease, Salla disease/Sialic AcidStorage Disease, Tay-Sachs/GM2 gangliosidosis, Wolman disease.

In further embodiments, use of the methods and materials is indicatedfor treatment of nervous system disease such as Rett Syndrome,Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, or fortreatment of nervous system injury including spinal cord and braintrauma/injury, stroke, and brain cancers.

In another aspect, the invention provides rAAV genomes. The rAAV genomescomprise one or more AAV ITRs flanking a polynucleotide encoding apolypeptide (including, but not limited to, an SMN polypeptide) orencoding siRNA, shRNA, antisense, and/or miRNA directed at mutatedproteins or control sequences of their genes. The polynucleotide isoperatively linked to transcriptional control DNAs, specificallypromoter DNA and polyadenylation signal sequence DNA that are functionalin target cells to form a gene cassette. The gene cassette may alsoinclude intron sequences to facilitate processing of an RNA transcriptwhen expressed in mammalian cells.

In some embodiments, the rAAV9 genome encodes a trophic or protectivefactor for treatment of neurodegenerative disorders, including but notlimited to Alzheimer's Disease, Parkinson's Disease, Huntington'sDisease along with nervous system injury including spinal cord and braintrauma/injury, stroke, and brain cancers. Non-limiting examples of knownnervous system growth factors include nerve growth factor (NGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophicfactor (CNTF), glial cell line-derived neurotrophic factor (GDNF), thefibroblast growth factor family (e.g., FGF's 1-15), leukemia inhibitoryfactor (LIF), certain members of the insulin-like growth factor family(e.g., IGF-1), the neurturins, persephin, the bone morphogenic proteins(BMPs), the immunophilins, the transforming growth factor (TGF) familyof growth factors, the neuregulins, epidermal growth factor (EGF),platelet-derived growth factor (PDGF), vascular endothelial growthfactor family (e.g. VEGF 165), follistatin, Hif1, and others. Alsogenerally contemplated are zinc finger transcription factors thatregulate each of the trophic or protective factors contemplated herein.In further embodiments, methods to modulate neuro-immune function arecontemplated, including but not limited to, inhibition of microglial andastroglial activation through, for example, NFkB inhibition, or NFkB forneuroprotection (dual action of NFkB and associated pathways indifferent cell types) by siRNA, shRNA, antisense, or miRNA. In stillfurther embodiments, the rAAV9 genome encodes an apoptotic inhibitor(e.g., bc12, bc1xL). Use of a rAAV9 encoding a trophic factor or spinalcord injury modulating protein or a suppressor of an inhibitor of axonalgrowth (e.g., a suppressor of Nogo [Oertle et al., The Journal ofNeuroscience, 23(13):5393-5406 (2003)] is also contemplated for treatingspinal cord injury.

For treatment of neurodegenerative disorders such as Parkinson'sdisease, the rAAV9 genome encodes in various embodiments Aromatic aciddopa decarboxylase (AADC), Tyrosine hydroxylase, GTP-cyclohydrolase 1(gtpch1), apoptotic inhibitors (e.g., bc12, bc1xL), glial cellline-derived neurotrophic factor (GDNF), the inhibitoryneurotransmitter-amino butyric acid (GABA), or enzymes involved indopamine biosynthesis. In further embodiments, the rAAV9 genome mayencode, for example, modifiers of Parkin and/or synuclein.

For treatment of neurodegenerative disorders such as Alzheimer'sdisease, in some embodiments, methods to increase acetylcholineproduction are contemplated. In some embodiments, methods of increasingthe level of a choline acetyltransferase (ChAT) or inhibiting theactivity of an acetylcholine esterase (AchE) are contemplated.

The rAAV9 genome encodes in some embodiments, siRNA, shRNA, antisense,and/or miRNA for use in methods to decrease mutant Huntington protein(htt) expression for treating a neurodegenerative disorder such asHuntington's disease.

The rAAV9 genome encodes in various embodiments siRNA, shRNA, antisense,and/or miRNA for use in for treatment of neurodegenerative disorderssuch as ALS. Treatment results in a decrease in the expression ofmolecular markers of disease, such as TNFα, nitric oxide, peroxynitrite,and/or nitric oxide synthase (NOS).

In some embodiments, the vectors encode short hairpin RNAs directed atmutated proteins such as superoxide dismutase for ALS, or neurotrophicfactors such as GDNF or IGF1 for ALS or Parkinson's disease.

In some embodiments, use of materials and methods of the invention isindicated for treating neurodevelopmental disorders such as RettSyndrome. For embodiments relating to Rett Syndrome, the rAAV9 genomemay encode, for example, methyl cytosine binding protein 2 (MeCP2).

“Treatment” comprises the step of administering via the intrathecalroute an effective dose, or effective multiple doses, of a compositioncomprising a rAAV of the invention to an animal (including a humanbeing) in need thereof. If the dose is administered prior to developmentof a disorder/disease, the administration is prophylactic. If the doseis administered after the development of a disorder/disease, theadministration is therapeutic. In embodiments of the invention, aneffective dose is a dose that alleviates (either eliminates or reduces)at least one symptom associated with the disorder/disease state beingtreated, that slows or prevents progression to a disorder/disease state,that slows or prevents progression of a disorder/disease state, thatdiminishes the extent of disease, that results in remission (partial ortotal) of disease, and/or that prolongs survival. Examples of diseasestates contemplated for treatment by methods of the invention are setout above.

Combination therapies are also contemplated by the invention.Combination as used herein includes both simultaneous treatment orsequential treatments. Combinations of methods of the invention withstandard medical treatments (e.g., riluzole in ALS) are specificallycontemplated, as are combinations with novel therapies.

While delivery to an individual in need thereof after birth iscontemplated, intrauteral delivery to a fetus is also contemplated.

Transduction with rAAV may also be carried out in vitro. In oneembodiment, desired target cells are removed from the subject,transduced with rAAV and reintroduced into the subject. Alternatively,syngeneic or xenogeneic cells can be used where those cells will notgenerate an inappropriate immune response in the subject.

Suitable methods for the transduction and reintroduction of transducedcells into a subject are known in the art. In one embodiment, cells canbe transduced in vitro by combining rAAV with the cells, e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest using conventional techniques such as Southern blots and/orPCR, or by using selectable markers. Transduced cells can then beformulated into pharmaceutical compositions, and the compositionintroduced into the subject by various techniques, such as by injectioninto the spinal cord.

The rAAV genomes of the invention lack AAV rep and cap DNA. AAV DNA inthe rAAV genomes (e.g., ITRs) may be from any AAV serotype for which arecombinant virus can be derived including, but not limited to, AAVserotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9,AAV-10 and AAV-11. The nucleotide sequences of the genomes of the AAVserotypes are known in the art. For example, the complete genome ofAAV-1 is provided in GenBank Accession No. NC_002077; the completegenome of AAV-2 is provided in GenBank Accession No. NC_001401 andSrivastava et al., J. Virol., 45: 555-564 {1983): the complete genome ofAAV-3 is provided in GenBank Accession No. NC_1829; the complete genomeof AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5genome is provided in GenBank Accession No. AF085716; the completegenome of AAV-6 is provided in GenBank Accession No. NC_00 1862; atleast portions of AAV-7 and AAV-8 genomes are provided in GenBankAccession Nos. AX753246 and AX753249, respectively; the AAV-9 genome isprovided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11genome is provided in Virology, 330(2): 375-383 (2004).

In another aspect, the invention provides DNA plasmids comprising rAAVgenomes of the invention. The DNA plasmids are transferred to cellspermissible for infection with a helper virus of AAV (e.g., adenovirus,E1-deleted adenovirus or herpesvirus) for assembly of the rAAV genomeinto infectious viral particles with AAV9 capsid proteins. Techniques toproduce rAAV particles, in which an AAV genome to be packaged, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV requires that the followingcomponents are present within a single cell (denoted herein as apackaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. Productionof pseudotyped rAAV is disclosed in, for example, WO 01/83692 which isincorporated by reference herein in its entirety. In variousembodiments, AAV capsid proteins may be modified to enhance delivery ofthe recombinant vector. Modifications to capsid proteins are generallyknown in the art. See, for example, US 2005/0053922 and US 2009/0202490,the disclosures of which are incorporated by reference herein in theirentirety.

A method of generating a packaging cell is to create a cell line thatstably expresses all the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes. AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line is then infected with a helpervirus such as adenovirus. The advantages of this method are that thecells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus rather than plasmids to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. No. 5,786,211; U.S. Pat.No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents arehereby incorporated by reference in their entirety herein, withparticular emphasis on those sections of the documents relating to rAAVproduction.

The invention thus provides packaging cells that produce infectiousrAAV. In one embodiment packaging cells may be stably transformed cancercells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293line). In another embodiment, packaging cells are cells that are nottransformed cancer cells such as low passage 293 cells (human fetalkidney cells transformed with El of adenovirus), MRC-5 cells (humanfetal fibroblasts). WI-38 cells (human fetal fibroblasts), Vero cells(monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

In other embodiments, the invention provides rAAV9 (i.e., infectiousencapsidated rAAV9 particles) comprising a rAAV genome of the invention.In one aspect of the invention, the rAAV genome is a self-complementarygenome.

In another aspect, rAAV are provided such as a rAAV9 named “rAAV SMN.”The rAAV SMN genome (nucleotides 980-3336 of SEQ ID NO: 1) has insequence an AAV2 ITR, the chicken β-actin promoter with acytomegalovirus enhancer, an SV40 intron, the SMN coding DNA set out in(GenBank Accession Number NM_000344.2), a polyadenylation signalsequence from bovine growth hormone and another AAV2 ITR. Conservativenucleotide substitutions of SMN DNA are also contemplated (e.g., aguanine to adenine change at position 625 of GenBank Accession NumberNM_000344.2). The genome lacks AAV rep and cap DNA, that is, there is noAAV rep or cap DNA between the ITRs of the genome. SMN polypeptidescontemplated include, but are not limited to, the human SMN1 polypeptideset out in NCBI protein database number NP_000335.1. Also contemplatedis the SMN1-modifier polypeptide plastin-3 (PLS3) [Oprea et al., Science320(5875): 524-527 (2008)]. Sequences encoding other polypeptides may besubstituted for the SMN DNA.

The rAAV may be purified by methods standard in the art such as bycolumn chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors from helper virus are known in the art andinclude methods disclosed in, for example, Clark et al., Hum. GeneTher., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med.,69: 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

In another aspect, the invention contemplates compositions comprisingrAAV of the present invention. In one embodiment, compositions of theinvention comprise a rAAV encoding a SMN polypeptide. In otherembodiments, compositions of the present invention may include two ormore rAAV encoding different polypeptides of interest.

Compositions of the invention comprise rAAV in a pharmaceuticallyacceptable carrier. The compositions may also comprise other ingredientssuch as diluents and adjuvants. Acceptable carriers, diluents andadjuvants are nontoxic to recipients and are preferably inert at thedosages and concentrations employed, and include buffers such asphosphate, citrate, or other organic acids; antioxidants such asascorbic acid; low molecular weight polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

Titers of rAAV to be administered in methods of the invention will varydepending, for example, on the particular rAAV, the mode ofadministration, the treatment goal, the individual, and the cell type(s)being targeted, and may be determined by methods standard in the art.Titers of rAAV may range from about 1×10⁶, about 1×10⁷, about 1×10⁸,about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, about 1×10¹², about 1×10¹³ toabout 1×10¹⁴ or more DNase resistant particles (DRP) per ml. Dosages mayalso be expressed in units of viral genomes (vg). Dosages may also varybased on the timing of the administration to a human. These dosages ofrAAV may range from about 1×10¹¹ vg/kg, about 1×10¹², about 1×10¹³,about 1×10¹⁴, about 1×10¹⁵, about 1×10¹⁶ or more viral genomes perkilogram body weight in an adult. For a neonate, the dosages of rAAV mayrange from about 1×10¹¹, about 1×10¹², about 3×10¹², about 1×10¹³, about3×10¹³, about 1×10¹⁴, about 3×10¹⁴, about 1×10¹⁵, about 3×10¹⁵, about1×10¹⁶, about 3×10¹⁶ or more viral genomes per kilogram body weight.

In another aspect, methods of transducing target cells (including, butnot limited to, nerve or glial cells) with rAAV are contemplated by theinvention.

The term “transduction” is used to refer to the administration/deliveryof a polynucleotide to a target cell either in vivo or in vitro, via areplication-deficient rAAV of the invention resulting in expression of afunctional polypeptide by the recipient cell.

Transduction of cells with rAAV of the invention results in sustainedexpression of polypeptide or RNA encoded by the rAAV. The presentinvention thus provides methods of administering/delivering rAAV (e.g.,encoding SMN protein) of the invention to an animal or a human patient.These methods include transducing nerve and/or glial cells with one ormore rAAV of the present invention. Transduction may be carried out withgene cassettes comprising tissue specific control elements. For example,promoters that allow expression specifically within neurons orspecifically within astrocytes. Examples include neuron specific enolaseand glial fibrillary acidic protein promoters. Inducible promoters underthe control of an ingested drug may also be developed.

In some aspects, it is contemplated that the transduction of cells isincreased when a vector of the disclosure is used in combination with acontrast agent as described herein relative to the transduction of avector of the disclosure when not used in combination with a contrastagent. In various embodiments, the transduction of cells is increased byat least about 1%, or at least about 5%, at least about 10%, at leastabout 20%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at leastabout 90%, at least about 100%, at least about 120%, at least about150%, at least about 180%, at least about 200%, at least about 250%, atleast about 300%, at least about 350%. at least about 400%, at leastabout 450%, at least about 500% or more when a vector of the disclosureis used in combination with a contrast agent as described herein,relative to the transduction of a vector of the disclosure when not usedin combination with a contrast agent. In further embodiments, thetransduction of cells is increased by about 10% to about 50%, or byabout 10% to about 100%, or by about 5% to about 10%, or by about 5% toabout 50%, or by about 1% to about 500%, or by about 10% to about 200%,or by about 10% to about 300%, or by about 10% to about 400%, or byabout 100% to about 500%, or by about 150% to about 300%, or by about200% to about 500% when a vector of the disclosure is used incombination with a contrast agent as described herein, relative to thetransduction of a vector of the disclosure when not used in combinationwith a contrast agent.

In some aspects, it is contemplated that the transduction of cells isfurther increased when a vector of the disclosure is used in combinationwith a contrast agent and when the patient is put in the Trendelenbergposition (head down position). In some embodiments, for example, thepatients is tilted in the head down position at about 1 degree to about30 degrees, about 15 to about 30 degrees, about 30 to about 60 degrees,about 60 to about 90 degrees, or about 90 up to about 180 degrees)during or after intrathecal vector infusion. In various embodiments, thetransduction of cells is increased by at least about 1%, or at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 100%, atleast about 120%, at least about 150%, at least about 180%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 450%, at least about 500% ormore when a vector of the disclosure is used in combination with acontrast agent and Trendelenberg position as described herein, relativeto the transduction of a vector of the disclosure when not used incombination with a contrast agent and Trendelenberg position. In furtherembodiments, the transduction of cells is increased by about 10% toabout 50%, or by about 10% to about 100%, or by about 5% to about 10%,or by about 5% to about 50%, or by about 1% to about 500%, or by about10% to about 200%, or by about 10% to about 300%, or by about 10% toabout 400%, or by about 100% to about 500%, or by about 150% to about300%, or by about 200% to about 500% when a vector of the disclosure isused in combination with a contrast agent and Trendelenberg position asdescribed herein, relative to the transduction of a vector of thedisclosure when not used in combination with a contrast agent andTrendelenberg position.

The disclosure also provides aspects wherein intrathecal administrationof a vector of the disclosure and a contrast agent to the centralnervous system of a patient in need thereof results in an increase insurvival of the patient relative to survival of the patient when avector of the disclosure is administered in the absence of the contrastagent. In various embodiments, administration of a vector of thedisclosure and a contrast agent to the central nervous system of apatient in need thereof results in an increase of survival of thepatient of at least about 1%, at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 100%, at least about 150%, at leastabout 200% or more relative to survival of the patient when a vector ofthe disclosure is administered in the absence of the contrast agent.

The disclosure also provides aspects wherein intrathecal administrationof a vector of the disclosure and a contrast agent to the centralnervous system of a patient in need thereof put in the Trendelenbergposition results in a further increase in survival of the patientrelative to survival of the patient when a vector of the disclosure isadministered in the absence of the contrast agent and the Trendelenbergposition. In various embodiments, administration of a vector of thedisclosure and a contrast agent to the central nervous system of apatient in need thereof put in the Trendelberg position results in anincrease of survival of the patient of at least about 1%, at least about5%, at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 100%, at leastabout 150%, at least about 200% or more relative to survival of thepatient when a vector of the disclosure is administered in the absenceof the contrast agent and the Trendelenberg position.

It will be understood by one of ordinary skill in the art that apolynucleotide delivered using the materials and methods of theinvention can be placed under regulatory control using systems known inthe art. By way of non-limiting example, it is understood that systemssuch as the tetracycline (TET on/off) system [see, for example, Urlingeret al., Proc. Nail. Acad. Sci. USA 97(14):7963-7968 (2000) for recentimprovements to the TET system] and Ecdysone receptor regulatable system[Palli et al., Eur J. Biochem 270: 1308-1315 (2003] may be utilized toprovide inducible polynucleotide expression. It will also be understoodby the skilled artisan that combinations of any of the methods andmaterials contemplated herein may be used for treating aneurodegenerative disease.

The present invention is illustrated by the following examples, whereinExample 1 describes the production of an exemplary rAAV9, Example 2describes the intrathecal administration of rAAV9, Example 3 describesthe increase in survival of SMN mutant mice followingintracerebroventricular (ICV) injection of rAAV9 SMN with contrast agentand Example 4 describes motor neuron transduction with a rAAV9 incynomologus monkeys.

EXAMPLE 1

The ability of rAAV9 to target and express protein in the centralnervous system was evaluated in an in vivo model system. The rAAV genomeincluded in sequence an AAV2 ITR, the chicken β-actin promoter with acytomegalovirus enhancer, an SV40 intron, green fluorescent protein(GFP) DNA, a polyadenylation signal sequence from bovine growth hormoneand another AAV2 ITR, as previously described in Bevan et al., MolecularTherapy, 19(11): 1971-1980 (2011).

Self-complementary AAV9 (AAV9 GFP) was produced by transienttransfection procedures using a double-stranded AAV2-ITR-based CB-GFPvector, with a plasmid encoding Rep2Cap9 sequence as previouslydescribed [Gao et al., J. Virol., 78: 6381-6388 (2004)] along with anadenoviral helper plasmid pHelper (Stratagene, Santa Clara, Calif.) in293 cells. The serotype 9 sequence was verified by sequencing and wasidentical to that previously described. Virus was produced in threeseparate batches for the experiments and purified by two cesium chloridedensity gradient purification steps, dialyzed against PBS and formulatedwith 0.001% Pluronic-F68 to prevent virus aggregation and stored at 4°C. All vector preparations were titered by quantitative PCR usingTaq-Man technology. Purity of vectors was assessed by 4-12% sodiumdodecyl sulfate-acrylamide gel electrophoresis and silver staining(Invitrogen, Carlsbad, Calif.).

EXAMPLE 2

Although some neurological disorders are caused by defects inubiquitously expressed proteins, in other disorders gene expression inthe CNS alone may have a substantial impact. The invention contemplatesthat gene delivery to the CSF can produce transduction along theneuraxis with the added benefit of potentially lowering the requireddose. Thus, to effect more localized CNS delivery, intrathecal and/orintracisternal injections of 5.2×10 12 vg/kg of AAV9 GFP and anon-ionic, low-osmolar contrast agent into 5-day-old pigs (n=3 each)were performed, and their brains and spinal cords were examined for GFPexpression.

Intrathecal Injection. Farm-bred sows (Sus scrofa domestica) wereobtained from a regional farm. Five-day-old (P5) piglets received 0.5cc/kg ketamine induction anesthesia and then were maintained by maskinhalation of 5% isoflurane in oxygen. Body temperature,electrocardiogram, and respiratory rate were monitored throughout theprocedure. For lumbar puncture, piglets were placed prone and the spinewas flexed in order to widen the intervertebral spaces. Theanterior-superior iliac spines were palpated and a line connecting thetwo points was visualized. The intervertebral space rostral to this lineis ˜L5-L6. Intraoperative fluoroscopy confirmed rostral-caudal andmediolateral trajectories. Using sterile technique, a 25-gauge needleattached to a 1-ml syringe was inserted. Gentle negative pressure wasapplied to the syringe as the needle was passed until a clear flash ofCSF was visualized. For cisterna puncture, the head of the piglet wasflexed while maintaining the integrity of the airway. Fluoroscopy againconfirmed adequate trajectory. A 25-gauge needle was passed immediatelycaudal to the occipital bone, and a flash of clear CSF confirmed entryinto the cistern magna.

For vector or control delivery, the syringe was removed while the needlewas held in place. A second 1-cc syringe containing either viralsolution (5.2×10 12 vg/kg) or PBS was secured and the solution wasinjected into the intrathecal space at a slow and constant rate. Afterdelivery, ˜0.25 ml of sterile PBS was flushed through the spinal needleso as to ensure full delivery of reagent. An iohexol radioopaque agent[Omnipaque™ (iohexol,N,N′-Bis(2,3-dihydroxypropyl)-5-[N(2,3-dihydroxypropyl)-acetamido]-2,4,6-trioldo-isophthalamide),GE Healthcare, Waukesha, Wis.] and recording intrathecal spread withreal-time continuous fluoroscopy.

Perfusion and tissue-processing. All subjects were sacrificed between 21and 24 days post-injection. Subjects were deeply anesthetized byintramuscular injection of Telazol followed by Propofol. A midventralsternal thoracotomy was performed and a cannula was inserted in theaorta through the left ventricle. The right atrium was opened and 0.5-11of PBS was injected through the cannula by gravity flow, followed byperfusion with 11 of 4% paraformaldehyde in phosphate buffer (pH 7.4).Organs were removed and post-fixed 48 hours in 4% paraformaldehydebefore further processing for histological sectioning or storedlong-term in 0.1% NaN3 PBS solution.

Histology and microscopy. Spinal cord segments were embedded in 3%agarose before cutting into 40-μm horizontal sections using a LeicaVT1200 vibrating microtome (Leica Microsystems, Buffalo Grove, Ill.).Sections were transferred in Tris-buffered saline and stored at 4° C.until processing. Brains were cryoprotected by successive incubation in10, 20, and 30% sucrose solutions. Once sufficiently cryoprotected(having sunk in 30% sucrose solution), brains were frozen andwhole-mounted on a modified Leica SM 2000R sliding microtome (LeicaMicrosystems) in OCT (Tissue-Tek, Torrance, Calif.) and cut into 40-μmcoronal sections.

For immunofluorescent determination of cell types transduced, floatingsections were submerged in blocking solution (10% donkey serum, 1%Triton-X100 in Tris-buffered saline) for 1 hour followed by overnightincubation in primary antibody solution at 4° C. The following primaryantibodies were used in this study: Rabbit-anti-GFP (1:500; Invitrogen),goat-anti-ChAT (1:100; Millipore, Billerica, Mass.),guinea-pig-anti-GFAP (1:1,000; Advanced Immunochemical, Long Beach,Calif.) and rabbit-anti-Iba1 (1:500; Dako, Carpentaria, Calif.). Primaryantibodies were detected using Fitc-, Cy3-, or Cy5-conjugated secondaryantibodies (1:1,000; Jackson ImmunoResearch, West Grove, Pa.) andmounted in PVA-DABCO medium.

For immunohistochemical staining, sections were incubated at roomtemperature in 0.5% H2O2/10% MeOH solution and subsequently blocked andstained as above with rabbit-anti-GFP overnight. Anti-GFP antibodieswere detected using biotinylated donkey-anti-rabbit secondary antibody(1:200; Jackson ImmunoResearch) and developed using Vector NovaRed perthe provided protocol (Vector Labs, Burlingame, Calif.). Sections werethen mounted in Cytoseal 60 medium (Thermo Fisher Scientific, Kalamazoo,Mich.).

Non-neural tissues were cut to ˜1 cm 3 blocks and cryoprotected byovernight incubation in 30% sucrose solution. They were then embedded ingum tragacanth and flash-frozen in liquid nitrogen-cooled isopentane.Samples were cut by cryostat into 10-12 μm sections and slides stored at−20° C. GFP expression was detected by a similar immunofluorescentprotocol as above with the addition of DAPI in secondary antibodysolution (1:1,000; Invitrogen).

Fluorescent images were captured using a Zeiss 710 Meta confocalmicroscope (Carl Zeiss MicroImaging, Thornwood, N.Y.) located at TRINCHand processed with LSM software.

Whole brain sections were scanned to x40 resolution at the BiopathologyCenter in the Research Informatics Core at the Research Institute atNationwide Children's Hospital using an Aperio automated slide scanner(Aperio, Vista, Calif.) and resulting images were processed withImageScope software.

In all animals, GFP expression was seen in the dorsal root ganglia aswell as the spinal cord gray and white matter. Importantly, AAV9 GFPinjection into either the cisternal space at the base of the skull orthe intrathecal space at L5 resulted in extensive motor neurontransduction and glia at all levels of the spinal cord as examined by insitu hybridization. Large ventral horn neurons were also positive forGFP expression by immunohistochemistry at all levels of spinal cord.Immunofluorescence confirmed that the GFP+cells expressed the motorneuron marker ChAT.

Finally, to further characterize the pattern of expression followingcisternal or intrathecal injection of AAV9-GFP into 5-day-old pigs,brains were examined for transgene expression again using GFPimmunofluorescence The regions with the highest levels of GFP expressionwere cerebellar Purkinje cells, nerve fibers within the medulla as wellas discrete nuclei, such as the olivary nucleus. Expression within therest of the brain was restricted to scattered cells near the meningealsurfaces. Examination of GFP expression in peripheral organs yielded novisible GFP expression indicating that the majority of the virus waslocalized to the CNS.

Thus, AAV9 injection into the cerebral spinal fluid of young pigsefficiently targeted motor neurons.

EXAMPLE 3

The effects of in vivo delivery of rAAV9 SMN [see Foust et al., NatureBiotechnology 28(3): 271-274 (2010) and description hereinabove, whereinthe sequence of the vector genome insert is shown as nucleotides980-3336 of SEQ ID NO: 1)] and contrast agent to the cerebral spinalfluid (CSF) of SMN mutant mice was tested.

Briefly, the rAAV9 SMN was mixed with contrast agent, followed by ICVinjection to effect placement of the composition to the CSF of SMNmutant mice. As a control experiment, the rAAV9 SMN vector was injectedwithout contrast agent into a separate group of SMN mutant mice.

Results showed that injection of rAAV9 SMN at ˜10⁸ vg/kg with contrastagent yielded a median survival of SMN mutant mice of 20 days, whileinjection of an equivalent amount of rAAV9 SMN in the absence ofcontrast agent yielded no survival.

Injection of rAAV9 SMN at ˜10⁹ vg/kg with contrast agent yielded amedian survival of SMN mutant mice of over 70 days, versus no survivalof SMN mutant mice that were injected with an equivalent amount of rAAV9SMN in the absence of contrast agent.

Finally, injection of rAAV9 SMN at ˜10¹⁰ vg/kg with contrast agentyielded a median survival of SMN mutant mice of over 100 days, versus amedian survival of 70 days in SMN mutant mice that were injected with anequivalent amount of rAAV9 SMN in the absence of contrast agent.

Thus, the survival of SMN mutant mice is increased following injectionof rAAV9 SMN with contrast agent, relative to the survival of SMN mutantmice following injection of rAAV9 SMN in the absence of contrast agent.

EXAMPLE 4

Three one year old cynomolgus monkeys received intrathecal injections of1×10¹³ vg/Kg rAAV9 encoding a shRNA and GFP. The injection was performedby lumbar puncture into the subarachnoid space of the lumbar thecal sac.The rAAV9 was resuspended with omnipaque (iohexol), an iodinatedcompound routinely used in the clinical setting. Iohexol is used tovalidate successful subarachnoid space cannulation and was administeredat a dose of 100 mg/Kg. The subject was placed in the lateral decubitusposition and the posterior midline injection site at ˜L4/5 levelidentified (below the conus of the spinal cord). Under sterileconditions, a spinal needle with stylet was inserted and subarachnoidcannulation was confirmed with the flow of clear CSF from the needle. Inorder to decrease the pressure in the subarachnoid space, 0.8 ml of CSFwas drained, immediately followed by injection with a mixture containing0.7 mL iohexol (300 mg/ml formulation) mixed with 2.1 mL of virus (2.8ml total). To investigate if rostral flow distribution of the viruscould improve cell transduction in the cervical area, one subject waslet recover in the lateral decubitus position, and the second and thirdsubjects were tilted in the Trendelenberg position (head down position).This is a routine procedure when performing CT myelograms in humansubjects.

Cynomolgus monkeys injected with virus were euthanized 2 weeks postinjection. Animals were anesthetized with sodium pentobarbital at thedose of 80-100 mg/kg intravenously and perfused with saline solution.Brain and spinal cord dissection were performed immediately and tissueswere processed either for nucleic acid isolation (snap frozen) orpost-fixed in 4% paraformaldehyde and subsequently cryoprotected with30% sucrose and frozen in isopentane at −65° C. 12 μm coronal sectionswere collected from lumbar cord using a cryostat for free floatingimmunostaining with green fluorescent protein (GFP) to identify thecells transduced by the virus and choline acetyl transferase (Chat) toidentify the motor neurons. Double positive cells were counted in 10sections of cervical, thoracic and lumbar cord and their number wasnormalized to the total number of Chat positive cells in the samesegment.

The cell counts revealed that tilting the subjects after virus infusionresults in a two-fold (100%) improvement in motor neuron transduction atthe thoracic and cervical levels.

While the present invention has been described in terms of variousembodiments and examples, it is understood that variations andimprovements will occur to those skilled in the art. Therefore, onlysuch limitations as appear in the claims should be placed on theinvention.

All documents referred to herein are incorporated by reference in theirentirety.

1. A composition comprising: (a) a recombinant AAV9 (rAAV9) comprisingan rAAV9 genome comprising a gene for treating a CLN disease selectedfrom the group consisting of a CLN1 gene, a CLN2 gene, a CLN3 gene, aCLN4 gene, a CLN5 gene, a CLN6 gene, and a CLN8 gene; and (b) contrastagent.
 2. The composition of claim 1, wherein the contrast agent is anon-ionic, low-osmolar contrast agent.
 3. The composition of claim 2,wherein the non-ionic, low-osmolar contrast agent is selected from thegroup consisting of iobitridol, iohexol, iomeprol, iopamidol, iopentol,iopromide, ioversol, ioxilan, and combinations thereof.
 4. Thecomposition of claim 2, wherein the non-ionic, low-osmolar contrastagent is iohexol.
 5. The composition of claim 1, wherein the rAAV9genome is a single-stranded genome.
 6. The composition of claim 1,wherein the rAAV9 genome is a self-complementary genome.
 7. Thecomposition of claim 1, wherein the gene for treating the CLN disease isa CLN1 gene.
 8. The composition of claim 1, wherein the gene fortreating the CLN disease is a CLN2 gene.
 9. The composition of claim 1,wherein the gene for treating the CLN disease is a CLN3 gene.
 10. Thecomposition of claim 1, wherein the gene for treating the CLN disease isa CLN4 gene.
 11. The composition of claim 1, wherein the gene fortreating the CLN disease is a CLN5 gene.
 12. The composition of claim 1,wherein the gene for treating the CLN disease is a CLN6 gene.
 13. Thecomposition of claim 1, wherein the gene for treating the CLN disease isa CLN8 gene.
 14. A method of treating a CLN disease in a patient in needthereof comprising, delivering a composition of claim 1 to a brain orspinal cord of a patient in need thereof, wherein the CLN disease is aCLN1, CLN2, CLN3, CLN4, CLN5, CLN6, or CLN8 disease.
 15. The method ofclaim 14, wherein the composition is delivered by intrathecal injection,intracisternal, or intracerebroventricular injection.
 16. The method ofclaim 15, further comprising placing the patient in the Trendelenbergposition after intrathecal injection of the composition.
 17. The methodof claim 14, wherein the contrast agent is a non-ionic, low-osmolarcontrast agent.
 18. The method of claim 17, wherein the non-ionic,low-osmolar contrast agent is selected from the group consisting ofiobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol,ioxilan, and combinations thereof.
 19. The method of claim 17, whereinthe non-ionic, low-osmolar contrast agent is iohexol.
 20. The method ofclaim 14, wherein the CLN disease is a CLN1 disease, and the gene fortreating the CLN disease is a CLN1 gene.
 21. The method of claim 14,wherein the CLN disease is a CLN2 disease, and the gene for treating theCLN disease is a CLN2 gene.
 22. The method of claim 14, wherein the CLNdisease is a CLN3 disease, and the gene for treating the CLN disease isa CLN3 gene.
 23. The method of claim 14, wherein the CLN disease is aCLN4 disease, and the gene for treating a CLN disease is a CLN4 gene.24. The method of claim 14, wherein the CLN disease is a CLN5 disease,and the gene for treating the CLN disease is a CLN5 gene.
 25. The methodof claim 14, wherein the CLN disease is a CLN6 disease, and the gene fortreating the CLN disease is a CLN6 gene.
 26. The method of claim 14,wherein the CLN disease is a CLN8 disease, and the gene for treating theCLN disease is a CLN8 gene.
 27. The method of claim 14, wherein thedelivering to the brain or spinal cord comprises delivery to a brainstem.
 28. The method of claim 14, wherein the delivering to the brain orspinal cord comprises delivery to a motor cortex.
 29. The method ofclaim 14, wherein the delivering to the brain or spinal cord comprisesdelivery to a nerve cell, a glial cell, or both.
 30. The method of claim14, wherein the delivering to the brain or spinal cord comprisesdelivery to a neuron, a lower motor neuron, a microglial cell, anoligodendrocyte, an astrocyte, a Schwann cell or combination thereof.